The disclosed invention is generally in the field of assays for detection of nucleic acids, and specifically in the field of nucleic acid amplification and mutation detection.
A number of methods have been developed which permit the implementation of extremely sensitive diagnostic assays based on nucleic acid detection. Most of these methods employ exponential amplification of targets or probes. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3 SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q.beta. replicase (Birkenneyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics, 9:199-202 (1993)).
While all of these methods offer good sensitivity, with a practical limit of detection of about 100 target molecules, all of them suffer from relatively low precision in quantitative measurements. This lack of precision manifests itself most dramatically when the diagnostic assay is implemented in multiplex format, that is, in a format designed for the simultaneous detection of several different target sequences.
In practical diagnostic applications it is desirable to assay for many targets simultaneously. Such multiplex assays are typically used to detect five or more targets. It is also desirable to obtain accurate quantitative data for the targets in these assays. For example, it has been demonstrated that viremia can be correlated with disease status for viruses such as HIV-1 and hepatitis C (Lefrere et al., Br. J. Haematol., 82(2):467-471 (1992), Gunji et al., Int. J. Cancer, 52(5):726-730 (1992), Hagiwara et al., Hepatology, 17(4):545-550 (1993), Lu et al., J. Infect. Dis., 168(5):1165-8116 (1993), Piatak et al., Science, 259(5102):1749-1754 (1993), Gupta et al., Ninth International Conference on AIDS/Fourth STD World Congress, Jun. 7-11, 1993, Berlin, Germany, Saksela et al., Proc. Natl. Acad. Sci. USA, 91(3):1104-1108 (1994)). A method for accurately quantitating viral load would be useful.
In a multiplex assay, it is especially desirable that quantitative measurements of different targets accurately reflect the true ratio of the target sequences. However, the data obtained using multiplexed, exponential nucleic acid amplification methods is at best semi-quantitative. A number of factors are involved:
1. When a multiplex assay involves different priming events for different target sequences, the relative efficiency of these events may vary for different targets. This is due to the stability and structural differences between the various primers used. PA0 2. If the rates of product strand renaturation differ for different targets, the extent of competition with priming events will not be the same for all targets. PA0 3. For reactions involving multiple ligation events, such as LCR, there may be small but significant differences in the relative efficiency of ligation events for each target sequence. Since the ligation events are repeated many times, this effect is magnified. PA0 4. For reactions involving reverse transcription (3SR, NASBA) or klenow strand displacement (SDA), the extent of polymerization processivity may differ among different target sequences. PA0 5. For assays involving different replicatable RNA probes, the replication efficiency of each probe is usually not the same, and hence the probes compete unequally in replication reactions catalyzed by Q.beta. replicase. PA0 6. A relatively small difference in yield in one cycle of amplification results in a large difference in amplification yield after several cycles. For example, in a PCR reaction with 25 amplification cycles and a 10% difference in yield per cycle, that is, 2-fold versus 1.8-fold amplification per cycle, the yield would be 2.0.sup.25 =33,554,000 versus 1.8.sup.25 =2,408,800. The difference in overall yield after 25 cycles is 14-fold. After 30 cycles of amplification, the yield difference would be more than 20-fold.
A method for amplifying and detecting nucleic acid sequences based on the presence of a specific target sequence using rolling circle replication is described in PCT Application WO 97/19193 by Yale University. In this method, a single stranded circular DNA molecule is replicated in an isothermal, continuous reaction to produce a single linear DNA molecule with numerous tandem repeats of the complement of the sequence of the circular DNA molecule. Replication is dependent on the presence of a primer specific for the circular DNA molecule. In the method of WO 97/19193, the primer is coupled to a binding moiety, such as an oligonucleotide probe or an antibody, that can bind to a specific molecule, such as a nucleic acid sequence or a protein. By making the presence of the primer dependent on the presence of the specific molecule (that is, the analyte), replication of the circular DNA molecule, which requires the primer, is made dependent on the presence of the analyte. In this way, detection of the tandem repeat DNA is made a surrogate for the presence of the analyte. This method is not optimal for separate detection of closely related sequences, such as single-base mutations or alleles, since hybridization discrimination between traditional probes differing in a single nucleotide is difficult to achieve.
Current technologies for quantitative profiling of mRNA/cDNA expression levels in biological samples involve the use of either cDNA arrays (Schena et al., Proc. Natl Acad. Sci. USA, 91:10614-10619 (1994)) or high density oligonucleotide arrays (Lockhart et al, Nature Biotechnology, 14:1675-1680 (1996)). In the case of the cDNA arrays by Schena et al, the detection of a single molecular species in each element of the array requires the presence of at least 100,000 bound target molecules. In the case of the DNA chip arrays used by Lockhart et al, the detection limit for hybridized RNA is of the order of 2000 molecules.
Current technologies for detection of mutations in DNA include cloning and genetic screens, DNA sequencing (with or without cloning), Single Strand Conformational Polymorphism analysis (SSCP), Multiple Allele-Specific Detection Assay (MASDA), oligonucleotide arrays (DNA chips, such as Affymetrix), and ASO-PCR, or PCR plus genetic bit analysis with sequencing primers. Methods of detecting nucleotide sequences by ligating together two probes which hybridize to adjacent sequences in the target nucleic acid molecule are described in U.S. Pat. Nos. 4,883,750, 5,242,794, and 5,521,065, all to Whiteley et al. These methods do not involve replication or other amplification of the signal generated by ligation. Of all these methods, only cloning and genetic screens, or cloning followed by DNA sequencing are capable of detecting somatic mutations that may occur at a level of one DNA strand in 10,000 wild type strands. Estimates of the accumulation of point mutations at the HPRT locus in normal tissues of 70-year old individuals is of the order of 2.times.10.sup.-7 per nucleotide (Simpson, Adv. Cancer Res. 71:209-240 (1997)). In tumor cells, the frequency may be 10 to 1000 times higher, depending on growth conditions (Richards et al., Science 277:1523-1526 (1997)). Thus, measurements of somatic mutation frequency in very early stages of cancer implies measuring mutation rates of the order of 2.times.10.sup.-6 to 2.times.10.sup.-5. Such infrequent events are difficult to measure using previous technologies.
It would be desirable to measure mutation rates using automated procedures. DNA sequencing of cloned material is an option, but the cost of sequencing a million bases per patient sample is prohibitive with current technology. High density DNA "chips" (Lockhart et al., 1996) have been used for mutation analysis (Hacia et al., Nature Genetics 14:441-447 (1997)). While the DNA chip and cDNA array technologies are capable of detecting mutations in 250,000 DNA loci simultaneously, the detection of somatic mutant DNA strands that represent less than 2% of the total DNA strands at a given locus is not possible. This is because the signals are generated by the averaging of thousands of hybridization events, and the mutant signal gets lost by dilution and cross-hybridization noise.
Accordingly, there is a need for nucleic acid detection methods that are both sensitive and can more easily distinguish between closely related sequences.
It is therefore an object of the disclosed invention to provide a method of detecting nucleic acid sequences that can discriminate between closely related sequences.
It is another object of the disclosed invention to provide a method of determining the amount of specific target nucleic acid sequences present in a sample where the number of signals measured is proportional to the amount of a target sequence in a sample and where the ratio of signals measured for different target sequences substantially matches the ratio of the amount of the different target sequences present in the sample.
It is another object of the disclosed invention to provide a method of detecting and determining the amount of multiple specific target nucleic acid sequences in a single sample where the ratio of signals measured for different target nucleic acid sequences substantially matches the ratio of the amount of the different target nucleic acid sequences present in the sample.
It is another object of the disclosed invention to provide a method of detecting the presence of single copies of target nucleic acid sequences. It is another object of the disclosed invention to provide a method of detecting the presence of target nucleic acid sequences representing individual alleles of a target genetic element.
It is another object of the disclosed invention to provide a method for detecting, and determining the relative amounts of, multiple molecules of interest in a sample.